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. 1992 Jun 1;175(6):1501–1509. doi: 10.1084/jem.175.6.1501

Long-term erythropoiesis from constant numbers of CD34+ cells in serum- free cultures initiated with highly purified progenitor cells from human bone marrow

PMCID: PMC2119250  PMID: 1375263

Abstract

To directly study the biological properties of purified hematopoietic colony-forming cell precursors, cells with a CD34+ CD45RAlo CD71lo phenotype were purified from human bone marrow using density separation and fluorescence-activated cell sorting, and were cultured in serum- free culture medium supplemented with various cytokines. In the presence of interleukin 3 (IL-3), IL-6, erythropoietin, and mast cell growth factor (a c-kit ligand), cell numbers increased approximately 10(6)-fold over a period of 4 wk, and the percentage of cells that expressed transferrin receptors (CD71) increased from less than 0.1% at day 0 to greater than 99% at day 14. Interestingly, the absolute number of CD34+ CD71lo cells did not change during culture. When CD34+ CD71lo cells were sorted from expanded cultures and recultured, extensive cell production was repeated, again without significant changes in the absolute number of cells with the CD34+ CD71lo phenotype that were used to initiate the (sub)cultures. These results document that primitive hematopoietic cells can generate progeny without an apparent decrease in the size of a precursor cell pool.

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Selected References

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  1. Andrews R. G., Singer J. W., Bernstein I. D. Precursors of colony-forming cells in humans can be distinguished from colony-forming cells by expression of the CD33 and CD34 antigens and light scatter properties. J Exp Med. 1989 May 1;169(5):1721–1731. doi: 10.1084/jem.169.5.1721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bernstein I. D., Andrews R. G., Zsebo K. M. Recombinant human stem cell factor enhances the formation of colonies by CD34+ and CD34+lin- cells, and the generation of colony-forming cell progeny from CD34+lin- cells cultured with interleukin-3, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor. Blood. 1991 Jun 1;77(11):2316–2321. [PubMed] [Google Scholar]
  3. Bigbee W. L., Vanderlaan M., Fong S. S., Jensen R. H. Monoclonal antibodies specific for the M- and N-forms of human glycophorin A. Mol Immunol. 1983 Dec;20(12):1353–1362. doi: 10.1016/0161-5890(83)90166-9. [DOI] [PubMed] [Google Scholar]
  4. Brandt J., Srour E. F., van Besien K., Briddell R. A., Hoffman R. Cytokine-dependent long-term culture of highly enriched precursors of hematopoietic progenitor cells from human bone marrow. J Clin Invest. 1990 Sep;86(3):932–941. doi: 10.1172/JCI114795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Broxmeyer H. E., Lu L., Hangoc G., Cooper S., Hendrie P. C., Ledbetter J. A., Xiao M., Williams D. E., Shen F. W. CD45 cell surface antigens are linked to stimulation of early human myeloid progenitor cells by interleukin 3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), a GM-CSF/IL-3 fusion protein, and mast cell growth factor (a c-kit ligand). J Exp Med. 1991 Aug 1;174(2):447–458. doi: 10.1084/jem.174.2.447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Curtis B. M., Williams D. E., Broxmeyer H. E., Dunn J., Farrah T., Jeffery E., Clevenger W., deRoos P., Martin U., Friend D. Enhanced hematopoietic activity of a human granulocyte/macrophage colony-stimulating factor-interleukin 3 fusion protein. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5809–5813. doi: 10.1073/pnas.88.13.5809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Drouet X., Douay L., Giarratana M. C., Baillou C., Gorin N. C., Salmon C., Najman A. Human liquid bone marrow culture in serum-free medium. Br J Haematol. 1989 Oct;73(2):143–147. doi: 10.1111/j.1365-2141.1989.tb00243.x. [DOI] [PubMed] [Google Scholar]
  8. Eaves C. J., Cashman J. D., Kay R. J., Dougherty G. J., Otsuka T., Gaboury L. A., Hogge D. E., Lansdorp P. M., Eaves A. C., Humphries R. K. Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures. II. Analysis of positive and negative regulators produced by stromal cells within the adherent layer. Blood. 1991 Jul 1;78(1):110–117. [PubMed] [Google Scholar]
  9. Hogge D. E., Cashman J. D., Humphries R. K., Eaves C. J. Differential and synergistic effects of human granulocyte-macrophage colony-stimulating factor and human granulocyte colony-stimulating factor on hematopoiesis in human long-term marrow cultures. Blood. 1991 Feb 1;77(3):493–499. [PubMed] [Google Scholar]
  10. Huang E., Nocka K., Beier D. R., Chu T. Y., Buck J., Lahm H. W., Wellner D., Leder P., Besmer P. The hematopoietic growth factor KL is encoded by the Sl locus and is the ligand of the c-kit receptor, the gene product of the W locus. Cell. 1990 Oct 5;63(1):225–233. doi: 10.1016/0092-8674(90)90303-v. [DOI] [PubMed] [Google Scholar]
  11. Iscove N. N., Guilbert L. J., Weyman C. Complete replacement of serum in primary cultures of erythropoietin-dependent red cell precursors (CFU-E) by albumin, transferrin, iron, unsaturated fatty acid, lecithin and cholesterol. Exp Cell Res. 1980 Mar;126(1):121–126. doi: 10.1016/0014-4827(80)90476-0. [DOI] [PubMed] [Google Scholar]
  12. Lansdorp P. M., Smith C., Safford M., Terstappen L. W., Thomas T. E. Single laser three color immunofluorescence staining procedures based on energy transfer between phycoerythrin and cyanine 5. Cytometry. 1991;12(8):723–730. doi: 10.1002/cyto.990120806. [DOI] [PubMed] [Google Scholar]
  13. Lansdorp P. M., Sutherland H. J., Eaves C. J. Selective expression of CD45 isoforms on functional subpopulations of CD34+ hemopoietic cells from human bone marrow. J Exp Med. 1990 Jul 1;172(1):363–366. doi: 10.1084/jem.172.1.363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Markowitz D., Goff S., Bank A. Construction and use of a safe and efficient amphotropic packaging cell line. Virology. 1988 Dec;167(2):400–406. [PubMed] [Google Scholar]
  15. Migliaccio G., Migliaccio A. R., Valinsky J., Langley K., Zsebo K., Visser J. W., Adamson J. W. Stem cell factor induces proliferation and differentiation of highly enriched murine hematopoietic cells. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7420–7424. doi: 10.1073/pnas.88.16.7420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Moore M. A. Review: Stratton Lecture 1990. Clinical implications of positive and negative hematopoietic stem cell regulators. Blood. 1991 Jul 1;78(1):1–19. [PubMed] [Google Scholar]
  17. Potten C. S., Hume W. J., Reid P., Cairns J. The segregation of DNA in epithelial stem cells. Cell. 1978 Nov;15(3):899–906. doi: 10.1016/0092-8674(78)90274-x. [DOI] [PubMed] [Google Scholar]
  18. Schmitt C., Eaves C. J., Lansdorp P. M. Expression of CD34 on human B cell precursors. Clin Exp Immunol. 1991 Jul;85(1):168–173. doi: 10.1111/j.1365-2249.1991.tb05699.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Southwick P. L., Ernst L. A., Tauriello E. W., Parker S. R., Mujumdar R. B., Mujumdar S. R., Clever H. A., Waggoner A. S. Cyanine dye labeling reagents--carboxymethylindocyanine succinimidyl esters. Cytometry. 1990;11(3):418–430. doi: 10.1002/cyto.990110313. [DOI] [PubMed] [Google Scholar]
  20. Sutherland H. J., Eaves C. J., Eaves A. C., Dragowska W., Lansdorp P. M. Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro. Blood. 1989 Oct;74(5):1563–1570. [PubMed] [Google Scholar]
  21. Sutherland H. J., Lansdorp P. M., Henkelman D. H., Eaves A. C., Eaves C. J. Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers. Proc Natl Acad Sci U S A. 1990 May;87(9):3584–3588. doi: 10.1073/pnas.87.9.3584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sutherland R., Delia D., Schneider C., Newman R., Kemshead J., Greaves M. Ubiquitous cell-surface glycoprotein on tumor cells is proliferation-associated receptor for transferrin. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4515–4519. doi: 10.1073/pnas.78.7.4515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Terstappen L. W., Huang S., Safford M., Lansdorp P. M., Loken M. R. Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD34+CD38- progenitor cells. Blood. 1991 Mar 15;77(6):1218–1227. [PubMed] [Google Scholar]
  24. Torok-Storb B. Cellular interactions. Blood. 1988 Aug;72(2):373–385. [PubMed] [Google Scholar]
  25. Udomsakdi C., Eaves C. J., Sutherland H. J., Lansdorp P. M. Separation of functionally distinct subpopulations of primitive human hematopoietic cells using rhodamine-123. Exp Hematol. 1991 Jun;19(5):338–342. [PubMed] [Google Scholar]
  26. Weilbaecher K., Weissman I., Blume K., Heimfeld S. Culture of phenotypically defined hematopoietic stem cells and other progenitors at limiting dilution on Dexter monolayers. Blood. 1991 Aug 15;78(4):945–952. [PubMed] [Google Scholar]
  27. Williams D. E., Eisenman J., Baird A., Rauch C., Van Ness K., March C. J., Park L. S., Martin U., Mochizuki D. Y., Boswell H. S. Identification of a ligand for the c-kit proto-oncogene. Cell. 1990 Oct 5;63(1):167–174. doi: 10.1016/0092-8674(90)90297-r. [DOI] [PubMed] [Google Scholar]
  28. Zsebo K. M., Wypych J., McNiece I. K., Lu H. S., Smith K. A., Karkare S. B., Sachdev R. K., Yuschenkoff V. N., Birkett N. C., Williams L. R. Identification, purification, and biological characterization of hematopoietic stem cell factor from buffalo rat liver--conditioned medium. Cell. 1990 Oct 5;63(1):195–201. doi: 10.1016/0092-8674(90)90300-4. [DOI] [PubMed] [Google Scholar]
  29. de Vries P., Brasel K. A., Eisenman J. R., Alpert A. R., Williams D. E. The effect of recombinant mast cell growth factor on purified murine hematopoietic stem cells. J Exp Med. 1991 May 1;173(5):1205–1211. doi: 10.1084/jem.173.5.1205. [DOI] [PMC free article] [PubMed] [Google Scholar]

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